Ayala-García Víctor M, Valenzuela-García Luz I, Setlow Peter, Pedraza-Reyes Mario
Department of Biology, University of Guanajuato, Guanajuato, Guanajuato, Mexico.
Department of Molecular Biology and Biophysics, UConn Health, Farmington, Connecticut, USA.
J Bacteriol. 2016 Nov 18;198(24):3345-3354. doi: 10.1128/JB.00625-16. Print 2016 Dec 15.
Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptional aag-lacZ fusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably, aag-lacZ expression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in a sigG-deficient background, suggesting a forespore-specific mechanism of aag transcription. Two additional findings supported this suggestion: (i) expression of an aag-yfp fusion was observed in the forespore, and (ii) in vivo mapping of the aag transcription start site revealed the existence of upstream regulatory sequences possessing homology to σ-dependent promoters. In comparison with the wild-type strain, disruption of aag significantly reduced survival of sporulating B. subtilis cells following nitrous acid or methyl methanesulfonate treatments, and the Rif mutation frequency was significantly increased in an aag strain. These results suggest that Aag protects the genome of developing B. subtilis sporangia from the cytotoxic and genotoxic effects of base deamination and alkylation.
In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.
枯草芽孢杆菌的Aag参与了体外从DNA中去除次黄嘌呤和烷基化碱基的过程。本文研究了枯草芽孢杆菌中aag的表达调控以及Aag缺陷菌株对遗传毒性剂的抗性和诱变特性。一个带有转录aag - lacZ融合基因的菌株在生长和早期孢子形成过程中表达低水平的β - 半乳糖苷酶，但在这个发育过程的后期阶段转录增加。值得注意的是，aag - lacZ在芽孢内的表达高于母细胞区室，并且在sigG缺陷背景下这种表达被消除，这表明aag转录存在芽孢特异性机制。另外两个发现支持了这一观点：（i）在芽孢中观察到aag - yfp融合基因的表达，（ii）aag转录起始位点的体内定位揭示了存在与σ依赖性启动子具有同源性的上游调控序列。与野生型菌株相比，aag的破坏显著降低了亚硝酸或甲磺酸甲酯处理后枯草芽孢杆菌孢子形成细胞的存活率，并且在aag菌株中利福平突变频率显著增加。这些结果表明Aag保护发育中的枯草芽孢杆菌孢子囊的基因组免受碱基脱氨基和烷基化的细胞毒性和遗传毒性作用。
在本研究中，有证据表明，编码参与次黄嘌呤和烷基化DNA碱基加工的DNA糖基化酶的aag，在枯草芽孢杆菌孢子形成过程中表现出芽孢特异性的基因表达模式。与这种时空表达模式一致，发现Aag保护这种微生物的孢子形成细胞免受碱基脱氨基和烷基化的有害和诱变作用。
